Acute Respiratory Distress Syndrome (ARDS) is a severe form of acute lung injury with very high mortality and few therapeutic options. Patients with acute lung injury are often initially treated with oxygen by mask, and if the disease progresses they will be mechanically ventilated with high levels of oxygen (typically 50 to 100%). Although both mechanical ventilation and hyperoxia (HO) can independently cause injury, the combination of the two has been shown to accelerate and cause more extensive lung injury than either condition alone. There is a critical need for understanding these mechanisms in order to develop strategies to reduce or prevent ventilator-induced lung injury (VILI). The long term goal is to improve understanding of the mechanisms by which the combination of exposure to high levels of oxygen (HO) and overdistention (or stretch) of lungs contributes to VILI. The overall objective of this application is to demonstrate that apoptosis signal regulating kinase-1 (ASK1) is a central mediator of HO-induced exacerbation of VILI. The central hypothesis of this application is that HO-induced activation of ASK1 promotes VILI by enhancing cell death through p38- mediated apoptosis and increased inflammation, and by preventing cell survival through suppression of extracellular signal-regulated kinase- (ERK1/2)-mediated pathways. These mechanisms will be investigated using ASK1-deficient mice in a clinically relevant mouse model with pre-exposure to HO (24 hr) followed by mechanical ventilation with high tidal volume. We will also investigate these mechanisms in mice with lung injury caused by LPS or influenza virus prior to exposure to HO and mechanical ventilation in order to model the clinical scenario of ARDS. In addition primary cultures of alveolar type II epithelial cells and macrophages, as well as cell lines with knockdown of ASK1 and other key mediators, will be subjected to HO and cyclic stretch. Aim 1 will test the hypothesis that HO/VILI is exacerbated by ASK1-induced activation of p38 that promotes stretch-induced apoptosis via inhibition of survivin and through activation of the inflammasome. Aim 2 will test the hypothesis that ASK1 activation prevents ERK1/2-mediated pro-survival pathways, including expression of survivin and Nrf2-mediated production of antioxidant enzymes. These studies will elucidate new signaling pathways involved in HO-induced exacerbation of VILI.